Pompe disease is a neuromuscular disorder resulting from mutations in the gene for acid ?-glucosidase (GAA) - an enzyme necessary to degrade lysosomal glycogen. Hypoventilation is a hallmark feature of Pompe disease and work from the first cycle of this grant demonstrated that neuropathology contributes to Pompe breathing problems. This is important since the current therapy for Pompe disease - intravenous enzyme replacement therapy (ERT) using recombinant GAA - does not target the central nervous system (CNS). This renewal application targets optimization of adeno-associated virus (AAV) based therapies to treat the CNS in Pompe disease. Aim 1 will use retrograde transport of AAV9 will be used to determine if gene therapy that selectively targets the entire motor unit (muscle and motoneuron) can correct a specific motor system. Specifically, Aim 1 will test the hypotheses that injection of AAV9 vector encoding the GAA gene (AAV9-GAA) into the tongue of Pompe (Gaa-/-) mice will cause GAA expression in muscle and motoneurons, and will restore tongue motor function. The hypoglossal motor system is being emphasized since it is impaired in Pompe disease with consequences to speech, swallow and breathing and does not respond to intravenous ERT. Aim 2 will focus on widespread CNS transduction by testing the hypotheses that intracisternal and intravenous AAV9-GAA delivery in Gaa-/- mice will transduce spinal cord and brainstem neurons, and will restore both tongue and diaphragm motor function. Aim 2 emphasizes the tongue and diaphragm since impaired breathing, ventilator-dependence and tongue motor problems are primary concerns in Pompe disease. Aim 3 is based on improving the ability of GAA to clear neuronal glycogen accumulation. We recently evaluated a modified form of recombinant GAA in which human GAA is fused to the ligand of the insulin-like growth factor II receptor (IGF-IIR). The resultant fusion protein has full catalytic activity for glycogenand shows an enhanced ability to reduce glycogen accumulation in our mouse model. Another modification of the GAA transgene will allow us to evaluate a highly conserved region of GAA which promotes processing to the most catalytically active 70kDa mature form of the enzyme. For the final aim we propose to package the gene for these enhanced forms of GAA into AAV9 to test the hypotheses that the modified GAA proteins more effectively target motoneurons. For all three aims a comprehensive series of outcome measures will be used to characterize respiratory function and AAV9 transduction. This work is a collaborative effort between a clinician and gene therapy researcher (B.J. Byrne) and a respiratory control scientist (D.D. Fuller). We believe this work is significant because the status quo in Pompe disease therapy is muscle-directed ERT. The substantial effort needed for bi-weekly ERT treatment, cost of >$500K per year, potential immune responses and limited success of ERT warrant an improved approach. The overall innovation of this work is that we are developing new AAV9 based therapies for respiratory insufficiency in Pompe disease. PUBLIC HEALTH RELEVANCE: Pompe disease is a lysosomal storage disorder associated with systemic deficiency of an enzyme (acid alpha glucosidase) which is required to degrade glycogen. We hypothesize that part of the reason that breathing problems are common in Pompe disease is that brainstem neurons and spinal cord motoneurons fail to adequately control the respiratory muscles. This renewal application targets optimization of adeno-associated virus (AAV) based therapies to treat respiratory neurons in Pompe disease.